This invention relates to novel cellulose-producing bacteria including one producing cellulose having high degrees of polymerization, one producing a Bingham polysaccharide as a by-product, and one producing a small amount of water-soluble polysaccharide; a method for the production of cellulosic material (bacterial cellulose: xe2x80x9cBCxe2x80x9d), which comprises culturing these cellulose-producing bacteria; and bacterial cellulose thus obtained.
Since the bacterial cellulose is edible as well as tasteless and odorless, it is utilized in the food industry. The homogenized bacterial cellulose""s high dispersibility in water further provides it with many industrial applications, such as to maintain particle sizes of food, cosmetics or coating agents, to strengthen food materials, to maintain moisture, to improve stability of food, and to be used as low-calorie additives and an emulsion stabilizer.
The bacterial cellulose is characterized by a sectional width of its fibrils which is smaller by two orders of magnitude than that of other kinds of cellulose such as those derived from wood pulp.
Due to such structural and physical feature of microfibril, a homogenized bacterial cellulose has plenty of industrial applications as a strengthening agent for polymers, especially hydrophilic polymers. Products prepared by solidification of the homogenized bacterial cellulose in the form of a lump or paper show a high elastic modulus in tension due to the above feature, and are therefore expected to have excellent mechanical properties for use in various kinds of industrial materials.
The methods for the production of BC are disclosed in Japanese Patent Laid-Open Application Sho 62(1987)-265990, Japanese Laid-Open Application Sho 63(1988)-202394 and Japanese Patent Application Publication Hei 6(1994)-43443.
Schramm/Hestrin medium is well known as a nutrient medium suitable for the cultivation of cellulose-producing bacteria, which comprises carbon sources, peptone, yeast-extract, sodium phosphate and citric acid (Schramm et al., J. General Biology, 11, pp.123-129, 1954).
It is also possible to optionally supply accelerators for the cellulose production such as inositol, phytic acid, pyrroloquinoline quinone (PQQ) (Japanese Patent Publication Hei 5(1993)-1718; Mitsuo TAKAI, Japan TAPPI Journal, Vol.42, No.3, pp.237-244), carboxylic acid or their salts (Japanese Patent Laid-Open Application Hei 7(1995)-39386, laid open Feb. 10, 1995), invertase (Japanese Patent Laid-Open Application Hei 7(1995)-184677, laid open Jul. 25, 1995) and methionine (Japanese Patent Laid-Open Application Hei 7(1995)-184675, laid open Sep. 25, 1995) into the culture media. Further, the BC production method with a cultivating apparatus having a specific oxygen-transfer coefficient (KLa) is disclosed in Japanese Patent Application Hei 7(1995)-31787.
Conventionally used culture conditions include static culture, shaken culture, and aerobic agitated culture, and conventionally used culture operation methods include batch fermentation, fed batch fermentation, repeated batch fermentation and continuous fermentation.
Means for agitation include impellers, air-lift fermenters, pump-driven recirculation of the fermenter broth and any combination of these means.
It is well known that the degrees of polymerization of BC are higher than wood pulp (e.g. LBKP and NBKP) and cotton linter used as industrial materials, but lower than a specific cellulose such as that derived from an ascidian and valonia. Polymeric materials including cellulose having higher degrees of polymerization will generally have more excellent mechanical properties such as strength and elasticity. Accordingly, it is expected that the cellulose derived from the ascidian and valonia having high degrees of polymerization is superior in the mechanical properties to the cellulose with low degrees of polymerization.
However, the above specific cellulose such as that derived from the ascidian or valonia has disadvantages that it exists little as resources and can not be efficiently collected, so that it will take much time to produce industrial materials from them. It has been therefore desired to industrially produce the BC having high degrees of polymerization.
Furthermore, it has been found that the BC produced by an industrially advantageous method such as those in an aerobic agitated culture has a lower weight-average degree of polymerization (DPw) than that produced in a static culture. Since the BC with lower degrees of polymerization has been deprived of the excellent mechanical properties, it has been desired to produce the BC having high degrees of polymerization by the aerobic agitated culture method.
It is also known that cellulose-producing bacteria producing no water-soluble polysaccharide as a by-product will show a high BC productivity in the static culture (Journal of General Microbiology (1988), 134, 1731-1736).
On the other hand, it is known that the by-product of water-soluble polysaccharide functions as dispersant in the agitated culture of the cellulose-producing bacteria. Adhesion or catching of BC in the parts of cultivation apparatuses such as impellers and stuffing of these parts with BC may be prevented by its functions so as to form the suspended BC into small clusters. As a result, the BC productivity will be increased (Japanese Patent Laid-Open Application Hei 5(1993)- 284988).
The present inventors have now found the bacteria capable of producing BC having high degrees of polymerization or BC containing only a small amount of the fraction with low degrees of polymerization even under the aerobic agitated culture condition, and the bacteria capable of producing only a small amount of water-soluble polysaccharide as the by-product so as to improve in their BC productivity and yield while maintaining the above function of the water-soluble polysaccharide. The present invention is based on these findings.
The present invention is related to cellulose-producing bacteria capable of producing of a bacterial cellulose having a weight-average degree of polymerization (in terms of polystyrene) of 1.6xc3x97104 or above, preferably of 1.7xc3x97104 or above, more preferably of 2.0xc3x97104 or above.
The weight-average degree of polymerization of a variety kinds of cellulose such as BC of this invention may be determined by the method using a GPC system (Tosoh HLC-8020) equipped with an RI detector as follows:
A cellulose sample is nitrated with a fuming nitric acid-phosphorous pentaoxide solution according to the method of W. J. Alexander, R. L. Mitchell, Analytical Chemistry 21, 12, 1497-1500 (1949).
Nitrated cotton linter is used as a control.
Nitrated cellulose is then dissolved in THF (Wako Pure Chemical Industries Ltd., the first grade) to a final concentration of 0.05%, and filtered through a 1.0 xcexcm pore-size filter. THF is also used for an elution solvent.
The flow rate, pressure, and sample-injection volume are adjusted to be 0.5 ml/min., 10-13 kgf/cm2 and 100 xcexcl, respectively.
The column system consists of two TSKgel GMH-HR (S) columns (7.5 IDxc3x97300 mm) and a guard column (Tosoh Co., Ltd.). The analysis is carried out at a temperature of 35xc2x0 C.
A relative molecular weight in terms of polystyrene is calculated by using polystyrene standards (Tosoh).
The polystyrene standards having a molecular weight in the range of 2.0xc3x97107 to 2630 are used and a standard curve is prepared based on the following three-dimension approximate equation:
logM=At3+Bt2+Ct+D
wherein xe2x80x9ctxe2x80x9d is an elution time and xe2x80x9cMxe2x80x9d is a molecular weight.
The weight-average molecular weight and number-average molecular weight are calculated by a program (ver. 3, 10) equipped in a data processor (SC-8020).
The weight-average degree of polymerization and number-average degree of polymerization of the original cellulose samples are finally calculated based on the above data, taking substitution degrees after the nitration into consideration.
The present invention is related to cellulose-producing bacteria producing a Bingham polysaccharide as the by-product.
The xe2x80x9cBingham polysaccharidexe2x80x9d means in this specification that an aqueous solution of the polysaccharide shows the same flow property as that of Bingham material, which may be determined by the method described herein below.
The Bingham material does not flow under the shear stress of a specific value (xcfx84f) or less, but will flow at a shear rate in proportion to the value (xcfx84-xcfx84f) when the shear stress is increased over the value (xcfx84f). The xe2x80x9cxcfx84fxe2x80x9d is called xe2x80x9cBingham yield valuexe2x80x9d.
The present invention is further related to cellulose-producing bacteria producing a small amount of water-soluble polysaccharide.
The xe2x80x9cproducing a small amount of water-soluble polysaccharidexe2x80x9d means that when the cellulose-producing bacteria are grown under the shaken flask culture conditions as described in Example 1, (3), they will produce a significant amount of less than about 2.1 g/l of water-soluble polysaccharide, or their yield against the consumed sugars (%) will reach less than about 6.7%, at the end of the culture.
Alternatively, it means that when the cellulose-producing bacteria are grown in a jar fermenter culture under the aerobic agitated conditions as described in Example 3, they will produce a significant amount of less than about 4.8 g/l of water-soluble polysaccharide, or their yield against the consumed sugars (%) will reach less than about 6.6%, at the end of the culture.
The amount of water-soluble polysaccharide is determined according to the method described in the present specification.
The present invention is also related to cellulose-producing bacteria capable of producing a bacterial cellulose containing such a small amount as less than about 24% by weight of the fraction with a degree of polymerization of 1,000 or less in terms of polystyrene (referred to hereinafter as the xe2x80x9cfraction with the low degrees of polymerizationxe2x80x9d) in the aerobic agitated culture.
The content of the fraction with the low degrees of polymerization is calculated from the % ratio of the area of the same fraction to the total area (100%) on a molecular weight distribution curve prepared by base-line correction of an elution curve obtained with the above data processor.
The present bacteria may be prepared by the treatment for mutagenesis with a known method using mutagens such as NTG (nitrosoguanidine) of Acetobacter strains such as Acetobacter xylinum subsp. sucrofermentans such as BPR 2001 strain, Acetobacter xylinum ATCC23768, Acetobacter xylinum ATCC23769, Acetobacter pasteurianus ATCC10245, Acetobacter xylinum ATCC14851, Acetobacter xylinum ATCC11142, Acetobacter xylinum ATCC10821; Agrobacterium; Rhizobium; Sarcina; Pseudomonas, Achromobacter; Alcaligenes; Aerobacter; Azotobacter; and Zooglea; followed by the isolation of the strains which have changed in shape and by the determination with the above GPC system of the weight-average degree of polymerization of the bacterial cellulose produced by the thus isolated mutants.
The BPR 2001 has been deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken 350 Japan) on Feb. 24, 1993 under accession number FERM P-13466, and then transferred on Feb. 7, 1994 to the deposit under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and Regulation under accession number FERM BP-4545.
The chemical mutagenesis treatment using the mutagens such as NTG is described in, for example, Japanese Patent Application Hei 6(1994)-127994, Bio Factors, Vol. 1, pp.297-302 (1988) and J. Gen. Microbiol, Vol. 135, pp.2917-2929 (1989). Accordingly, those skilled in the art may obtain the present mutants in accordance with these known methods. The present mutants may be also obtained by other treatments such as application of radioactive rays.
One example of the present cellulose-producing bacteria, BPR3001A, has been deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken 350 Japan) on Jun. 12, 1995 under accession number FERM P-14982, and then transferred on Feb. 23, 1996 to the deposit under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and Regulation under accession number FERM BP-5421.
The present invention also relates to a bacterial cellulose having a weight-average degree of polymerization in terms of polystyrene of 1.6xc3x97104 or above, preferably 1.7xc3x97104 or above, which may be produced in the aerobic agitated culture; a bacterial cellulose having a weight-average degree of polymerization in terms of polystyrene of 2.0xc3x97104 or above; and a bacterial cellulose containing less than about 24% by weight of the fraction with the degree of polymerization of 1,000 or less in terms of polystyrene, which may be produced in the aerobic agitated culture.
The present invention is finally related to a method for the production of bacterial cellulose, which comprises culturing these cellulose-producing bacteria; bacterial cellulose thus obtained; and shaped articles made of these bacterial cellulose.
The shaped articles according to the present invention include various industrial materials such as a sheet, fibre and film, which have excellent mechanical properties such as high strength and high elasticity.
Carbon sources in the culture media useful in the present invention include sucrose, glucose, fructose, mannitol, sorbitol, galactose, maltose, erythritol, glycerol, ethyleneglycol, ethanol and their mixtures. In addition, sucrose may be combined with starch hydrolysate containing these carbon sources, citrus molasses, beet molasses, squeezed juice from beet or sugar cane, juice from citrus and the like.
Nitrogen sources useful in the present invention include organic or inorganic ones such as ammonium salts including ammonium sulfate, ammonium chloride, ammonium phosphate; nitrates; and urea. Nitrogen-containing natural nutrients may be also used including Bacto-Peptone, Bacto-soytone, Yeast-Extract and Bean-Condensate.
A trace amount of organic nutrients may be further added including amino acids, vitamins, fatty acids, nucleic acids, 2,7,9-tricarboxy-1H pyrrolo [2,3,5]-quinoline-4,5-dione, sulfite pulp waste liquor, lignin sulfonic acid and the like.
When the mutants with the nutritional requirement for amino acids are used, such required nutrients should be supplemented in the culture media. Inorganic nutrients include phosphate salts, magnesium salts, calcium salts, iron salts, manganese salts, cobalt salts, molybdate salts, hematite salts, chelate metal salts and the like.
It is also possible to optionally supply the above-mentioned accelerators for the cellulose production into the culture media.
For example, when the Acetobacter is used as the cellulose-producing bacteria, a pH range for the culture is controlled between 3 and 7, preferably around 5. A culture temperature is kept in a range between 10 and 40xc2x0 C., preferably between 25 and 35xc2x0 C. Oxygen supply into a culturing apparatus may contain from 1 to 100% oxygen, desirably 21 to 80%. Those skilled in the art may optionally determine the contents of these components in the culture media and amounts of the bacteria to be inoculated into the media, depending on the culture method to be used.
The present method may be carried out in the known culture conditions such as the static culture and aerobic agitated culture, as described above.
Means for agitation include impellers, air-lift fermenters, pump-driven recirculation of the fermenter broth and any combination of these means.
Any known culture operation methods such as batch fermentation, fed batch fermentation, repeated batch fermentation and continuous fermentation may be adopted.
Further, the bacterial cellulose may be also produced by the method described in Japanese Patent Laid-Open Application Hei 8(1996)-33494 (laid open Feb. 6, 1996) in the name of the present applicant, wherein culture media containing bacteria are circulated between a culturing apparatus and a separator to separate the resulting bacterial cellulose from the bacteria and culture media in said separator, or by the method described in Japanese Patent Laid Open Application Hei 8(1996)-33495 (laid open Feb. 6, 1996) in the name of the present applicant, wherein the concentration of the bacterial cellulose in culture media is kept at a lower level by a continuous removal of the culture media from its culture system and a continuous supply of fresh culture media having almost the same volume as the removed culture media.
The aerobic agitated culture may be carried out in any culturing apparatus with agitation, such as a jar fermenter and tank.
In the present aerobic agitated culture, gasses may be optionally passed through the culture media. Such gasses include oxygen-containing gases such as air, as well as gasses free of oxygen such as argon or nitrogen. Those skilled in the art may optionally select the gas to be passed, depending on the culture conditions.
For example, when anaerobic bacteria are used, an inert gas may be passed through the culture media so that the bubbles thereof will agitate the culture media.
When aerobic bacteria are used, an oxygen-containing gas may be passed through the culture media to supply oxygen required for the growth of the bacteria. The bubbles thereof will also agitate the culture media.
The bacterial cellulose having less than about 24% by weight of the fraction with the degree of polymerization of 1,000 or less in terms of polystyrene may be produced by other methods than the aerobic agitated culture.
Thus, the bacterial cellulose with a small amount of the fraction of the low degrees of polymerization may be produced by culturing the cellulose-producing bacteria while controlling the activity of cellulose secreted by the same bacteria into the culture media.
For example, endo-cellulase (CMCase) secreted by the cellulose-producing strain, BPR2001, shows a relatively high activity at a pH range of from about 4.5 to about 5.5 with a maximum activity at pH 5. The activity will decrease at lower or higher pH ranges, showing about 10% activity of the maximum one at pH 4 or less. However, the enzyme will never be inactivated even at pH 4 or less.
Accordingly, one specific method for controlling the above cellulase activity may be maintaining the pH of a medium in a range of below about 4.5 or above about 5.5, preferably at about pH 4.
The cellulase activity may be controlled by other ways such as, for example, by limiting other medium conditions such as a salt concentration or by adding an appropriate amount of a reagent capable of inhibiting the cellulase activity into the culture medium.
Further, it is preferred to control the cellulose activity at BC purification and separation steps as well, since the remaining cellulase in the culture medium after the completion of the cultivation can decrease the degree of polymerization of BC. In such case, the cellulase activity may be controlled by any method such as pH limitation, addition of the cellulase inhibitor, heat treatment and alkaline treatment.
The activity of CMCase is determined on the basis of the decrease of viscosity according to J. Biol. Chem. Vol. 250, pp.1012-1018 (1975).
Thus, an enzyme sample (100 xcexcl) is added to 15 ml of a carboxylmethylcellulose (CMC) solution (0.6%), and the change of viscosity of the mixture is measured during the course of time with a viscometer (Vibro Viscometer CVJ-5000; Chichibu Cement Co. Ltd., Tokyo) at 30xc2x0 C. An amount of the enzyme which is necessary for causing 1% decrease of the viscosity in 2 h is defined as one activity unit (1 U).
The bacterial cellulose with a small amount of the fraction with the low degrees of polymerization may be produced by other production methods such as, for example, an aerobic agitated culture of the cellulose-producing bacteria with a culture medium containing no corn steep liquor (CSL), culturing the bacteria with water-soluble polysaccharide added in a culture medium and culturing the bacteria producing polysaccharide as the by-product.
The bacterial cellulose according to the present invention may be subjected to homogenization.
The homogenization of the bacterial cellulose is considered to be a phenomenon in which the cellulose is deformed and broken under a stress induced inside the cellulose by an external force such as a mechanical force. Accordingly, the homogenization of the bacterial cellulose may be carried out by externally applying a mechanical force to the bacterial cellulose.
The mechanical force includes tensile stress, bending stress, compressive stress, torsional stress, impact stress and shearing stress. Compressive stress, impact stress and shearing stress are generally dominating.
A practical application of these mechanical forces to the bacterial cellulose may be achieved by using an appropriate apparatus such as a cooking mixer, homogenizer, blender, Polytron or ultrasonic generator.
In the homogenization using the above apparatus, the mechanical force is mainly composed of the impact force generated from the collision between agitating blades and the bacterial cellulose, and of the shearing force generated due to the differences in the speeds of the suspension.
In the homogenization using Polytron, the mechanical force is mainly composed of the compressive force generated by sandwiching the bacterial cellulose between outer blades and inner blades, of the impact force generated from the collision between the bacterial cellulose and blades rotating at a high speed, and of the shearing stress generated in the suspension at a space between stopping outer blades and inner blades rotating at a high speed.
In the homogenization using the ultrasonic generator, the mechanical force is mainly composed of a strong shearing stress locally generated by a continuous cavitation in the suspension due to the oscillation of the ultrasonic generator.
In addition to the above embodiments, the present homogenization may be carried out in any manner for externally applying a certain load (mechanical force) to the bacterial cellulose.
The homogenized bacterial cellulose serving for an excellent emulsifier may be prepared by homogenization of a suspension containing about 1% or less by weight of bacterial cellulose. Those skilled in the art may optionally select the other homogenization conditions.
The bacterial cellulose may be recovered as such, or then impurities other than the bacterial cellulose, including the bacteria per se, may be removed from the recovered bacterial cellulose.